Nasopharyngeal Microbiota Profiling of Pregnant Women With Sars-Cov-2 Infection (preprint)

Background: We aimed to characterize the role of the nasopharyngeal microbiota in pregnant women with and without SARS-CoV-2 infection.Methods: Pregnant women were enrolled from a multicenter prospective population-based cohort (March-June 2020 in Barcelona, Spain) in which the status of SARS-CoV-2 infection was determined by nasopharyngeal RT–PCR and antibodies in peripheral blood. DNA was extracted from nasopharyngeal swab samples, and the V3-V4 region of the 16S rRNA of bacteria was amplified using region-specific primers. The differential abundance of taxa was tested, and alpha/beta diversity was evaluated.Results: Among 76 women, 38 were classified as positive and 38 as negative for SARS-CoV-2 infection. All positive women were diagnosed by antibodies, and 14 (37%) also had a positive RT–PCR. SARS-CoV-2 infection altered the overall composition of the nasopharyngeal microbiota (p=0.001), with a higher relative abundance of the Tenericutes and Bacteroidetes phyla and a higher abundance of the Prevotellaceae family. Infected women presented a different pattern of microbiota profiling due to beta diversity and higher richness (observed ASV<0.001) and evenness (Shannon index<0.001) at alpha diversity. These changes persisted after acute infection, as revealed by negative RT–PCR but positive antibodies, suggesting a long-lasting effect of SARS-CoV-2 in the nasopharyngeal microbiota. No significant differences were reported in mild vs. severe cases, suggesting a role of the SARS-CoV-2 infection itself but not on its severity.Conclusion: This is the first study on nasopharyngeal microbiota during pregnancy. SARS-CoV-2 infection altered the overall structure and diversity of the nasopharyngeal microbiota profile, and this effect seems to persist after the acute moment of the infection.

SARS-CoV-2 RNA and antibody detection in human milk from a prospective multicenter study in Spain (preprint)

BackgroundDuring the COVID-19 pandemic in 2020, breastfeeding in women positive for SARS-CoV-2 was compromised due to contradictory data regarding potential viral transmission. However, growing evidence confirms the relevant role of breast milk in providing passive immunity by generating and transmitting specific antibodies against the virus. Thus, our study aimed to develop and validate a specific protocol to detect SARS-CoV-2 in breast milk matrix as well as to determine the impact of maternal SARS-CoV-2 infection on presence, concentration, and persistence of specific SARS-CoV-2 antibodies. Study design/MethodsA prospective multicenter longitudinal study in Spain was carried out from April to December 2020. A total of 60 mothers with SARS-CoV-2 infection and/or recovered from COVID-19 were included (n=52 PCR-diagnosed and n=8 seropositive). Data from maternal-infant clinical records and symptomatology were collected. A specific protocol was validated to detect SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene. Presence and levels of SARS-CoV-2 specific immunoglobulins (Igs) -IgA, IgG, and IgM-in breast milk samples from COVID-19 patients and from 13 women before the pandemic were also evaluated. ResultsAll breast milk samples showed negative results for SARS-CoV-2 RNA presence. We observed high intra- and inter-individual variability in the antibody response to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Protease domain (MPro) antibodies were also detected in milk. In general, 82.9 % of the milk samples were positive for at least one of the three antibody isotypes, being 52.86 % of those positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2 - 87.5 %), whereas it raised continuously for IgG (47.8 % the first ten days to 87.5 % from day 41 up to day 206 post-PCR confirmation). ConclusionsConsidering the lack of evidence for SARS-CoV-2 transmission through breast milk, our study confirms the safety of breastfeeding practices and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer, that would provide passive immunity to breastfed infants and protect them against COVID-19 disease. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence.

Agreement between commercially available ELISA and in-house Luminex SARS-CoV-2 antibody immunoassays (preprint)

Serological diagnostic of the severe respiratory distress syndrome coronavirus 2 (SARS-CoV-2) is a valuable tool for the determination of immunity and surveillance of exposure to the virus. In the context of an ongoing pandemic, it is essential to externally validate widely used tests to assure correct diagnostics and epidemiological estimations. We evaluated the performance of the COVID-19 ELISA IgG and IgM/A (Vircell, S.L.) against a highly specific and sensitive in-house Luminex immunoassay in a set of samples from pregnant women and cord blood. The agreement between both assays was moderate to high for IgG but low for IgM/A. Considering seropositivity by either IgG and/or IgM/A, the technical performance of the ELISA was highly imbalanced, with 96% sensitivity at the expense of 22% specificity. As for the clinical performance, the negative predictive value reached 87% while the positive predictive value was 51%. Our results stress the need for highly specific and sensitive assays and external validation of diagnostic tests with different sets of samples to avoid the clinical, epidemiological and personal disturbances derived from serological misdiagnosis.

LOW BIRTH WEIGHT AS A RISK FACTOR FOR SEVERE COVID-19 IN ADULTS (preprint)

The identification of factors predisposing to severe COVID-19 in young adults remains partially characterized. Low birth weight (LBW) alters cardiovascular and lung development and predisposes to adult disease. We hypothesized that LBW is a risk factor for severe COVID-19 in non-elderly subjects. We analyzed a prospective cohort of 397 patients (18-70y) with laboratory-confirmed SARS-CoV-2 infection attended in a tertiary hospital, where 15% required admission to Intensive Care Unit (ICU). Perinatal and current potentially predictive variables were obtained from all patients and LBW was defined as birth weight [≤]2,500 g. Age (adjusted OR (aOR) 1.04 [1-1.07], P=0.012), male sex (aOR 3.39 [1.72-6.67], P<0.001), hypertension (aOR 3.37 [1.69-6.72], P=0.001), and LBW (aOR 3.61 [1.55-8.43], P=0.003) independently predicted admission to ICU. The area under the receiver-operating characteristics curve (AUC) of this model was 0.79 [95% CI, 0.74-0.85], with positive and negative predictive values of 29.1% and 97.6% respectively. Results were reproduced in an independent cohort, from a web-based survey in 1,822 subjects who self-reported laboratory-positive SARS-CoV-2 infection, where 46 patients (2.5%) needed ICU admission (AUC 0.74 [95% CI 0.68-0.81]). LBW seems to be an independent risk factor for severe COVID-19 in non-elderly adults and might improve the performance of risk stratification algorithms.

COVID-19 causing HELLP-like syndrome in pregnancy and role of angiogenic factors for differential diagnosis (preprint)

Importance: The clinical presentation of hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is one of the more severe forms of preeclampsia. COVID-19 infection exhibits signs that are shared with preeclampsia and HELLP syndrome, which may lead to needless interventions and iatrogenic preterm delivery. Objective: We evaluated the prevalence of HELLP-like signs in pregnant women admitted for COVID-19 and the value of angiogenic factors to rule out preeclampsia. Methods: a consecutive series of 27 pregnant women beyond 20 weeks of gestation, with symptomatic COVID-19. Clinical and analytical features were recorded and those cases with signs of HELLP syndrome were tested for sFlt-1/PlGF ratio. Results: Seven patients (25.9%) presented at least one sign of suspected HELLP syndrome, of which 2 (7.4%) were diagnosed clinically with PE because of hypertension and high transaminases and 5 (18.5%) had only elevated transaminases. sFlt-1/PlGF ratio was normal in 6 of 7. Conclusion: Symptomatic COVID-19 may simulate severe preeclampsia in pregnancy. Angiogenic factors may be essential to avoid false diagnosis and needless interventions. These data were presented in a Virtual Symposium on Covid-19 and Pregnancy on 17 April: 2020:(http://medicinafetalbarcelona.org/simposiocovid19/ [Spanish] and https://medicinafetalbarcelona.org/symposiumcovid19/ [English]

SEROPREVALENCE AND CLINICAL SPECTRUM OF SARS-CoV-2 INFECTION IN THE FIRST VERSUS THIRD TRIMESTER OF PREGNANCY (preprint)

Introduction: Case registries of pregnant women diagnosed with coronavirus disease (COVID-19) by polymerase chain reaction (PCR) have reported that the majority experienced mild infection, but up to 9% may require critical care. Most COVID-19 cases published were in the third trimester of pregnancy, which could reflect reporting bias, higher risk of infection or increased disease severity in late pregnancy. Seroprevalence studies may allow reliable estimates of the susceptibility to infection and clinical spectrum since they include asymptomatic and mild infections not tested for PCR. We evaluated the seroprevalence and clinical presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pregnant women in the first and third trimester. Methods: The study was approved by the Institutional Review Board at each institution and informed consent was obtained. We recruited 874 consecutive pregnancies attending for first trimester screening (10-16 weeks of gestation, n=372) or delivery (n=502) from April 14 to May 5. All women were interviewed with a structured questionnaire for COVID-19 symptoms two months prior to sampling. SARS-CoV-2 IgG and IgM/IgA antibodies were tested (COVID-19 VIRCLIA Monotest, Vircell Microbiologist, Spain; reported sensitivity 70% IgG and 89% IgM/IgA, and specificity 89% and 99% respectively). Indeterminate results were re-tested (VITROS Immunodiagnostic Products Anti-SARS-CoV2 Total Tests, Ortho Clinical Diagnostics, USA; 100% sensitivity and specificity) and re-classified as positive or negative. Women with COVID-19 were diagnosed and managed according to standard protocols and guidelines3,4. Statistical differences were tested using the {chi}2 test or Student t-test as appropriate (p<0.05). Results: A total of 125 of 874 women (14.3%) were positive for either IgG or IgM/IgA SARS-CoV-2 antibodies, 54/372 (14.5%) in the first and 71/502 (14.1%) in the third trimester. A total of 75/125 (60%) reported no symptoms of COVID-19 in the past 2 months, whereas 44 (35.2%) reported one or more symptoms, of which 31 (24.8%) had at least 3 symptoms or anosmia and 8 (6.4%) dyspnea. Overall, 7 women (5.6%) were admitted for persistent fever despite paracetamol and dyspnea, of which 3 had signs of pneumonia on chest radiography. All 3 had criteria for severity (bilateral chest condensation, respiratory rate>30 and leukopenia) and required oxygen support but not critical care or mechanical ventilation, and they were all discharged well. The rates of symptomatic infection, hospital admission or dyspnea were significantly higher in third trimester women (Table and Figure). Discussion: The 14.3% seroprevalence of SARS-COV-2 in pregnant women in this study was substantially larger than the contemporary rates of PCR positive cases (0.78%) reported for women 20-40y in Barcelona. The data confirm that COVID-19 is asymptomatic in the majority of pregnant women6 and illustrate the value of seroprevalence studies to capture the high proportion of asymptomatic or mild infections. In this study, none of the 125 pregnant women with SARS-CoV-2 infection required critical care as compared to 9% reported in cases diagnosed with PCR. However, the proportion of infections with symptoms or dyspnea was remarkably higher in the third trimester, and these results are in line with COVID-19 registries, reporting that 81% of hospitalized women were in late pregnancy or peripartum. These results provide reassuring information that, even in settings with a high prevalence, SARS-CoV-2 infection in pregnancy mostly presents with asymptomatic or mild clinical forms. The susceptibility to infection seemed to be the same in the first and the third trimesters of gestation. The data further suggest that, as with other respiratory viruses, COVID-19 could be more severe and require increased surveillance in late pregnancy. These findings should be confirmed and extended with larger consecutive prevalence studies in pregnancy.